The RGS (regulators of G-protein signalling) protein family sharpen signalling kinetics through heterotrimeric G-proteins by enhancing the GTPase activity of the G-protein α subunit. Paradoxically, they also accelerate receptor-stimulated activation. We investigated this paradox using the cloned G-protein gated K+ channel as a reporter of the G-protein cycle, and FRET (fluorescence resonance energy transfer) between cyan and yellow fluorescent protein tagged proteins to detect physical interactions. Our results with the neuronal protein, RGS8, show that the enhancement of activation kinetics is a variable phenomenon determined by receptor type, G-protein isoform and RGS8 expression levels. In contrast, deactivation was consistently accelerated after removal of agonist. FRET microscopy revealed a stable physical interaction between RGS8-yellow fluorescent protein and Go αA-cyan fluorescent protein that occurred in the presence and absence of receptor activation and was not competed away by Gβγ overexpression. FRET was also seen between RGS8 and Gγ, demonstrating that RGS8 binds to the heterotrimeric G-protein as well as G-protein α subunit-GTP and the transition complex. We propose a novel model for the action of RGS proteins on the G-protein cycle involving participation of the RGS in the ternary complex: for certain combinations of agonist, receptor and G-protein, RGS8 expression improves upon the ‘kinetic efficacy’ of G-protein activation.

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